PROJECT SUMMARY/ABSTRACT Vascular barrier dysfunction causes aberrant transport of blood components into the vessel wall or surrounding tissues, a hallmark of inflammatory injury in response to trauma, sepsis, atherosclerosis, diabetes, and stroke. Currently, there are no effective therapies that directly target the leaky barrier, as drug development has been hampered by knowledge gaps and difficulties in translating cell/animal data to human pathophysiology. Our program addresses these challenges via comparative analyses of endothelial barrier structure and function in human and animal models of inflammatory injury. We conduct three series of studies in the blood, blood-vessel interface and endothelial barrier structure, aimed at 1) identifying key circulating factors that cause barrier leakage and their cell-specific mechanisms of production and action; 2) characterizing endothelial surface receptors and intracellular signals that transduce their effects; and 3) elucidating molecular events in cell-cell junctions, cytoskeleton, and glycocalyx that ultimately lead to barrier opening. Our work has continuously been supported by the NHLBI contributing to the development of novel techniques and transformative theories in vascular permeability. We were among the first to characterize the nmMLCK signaling in endothelial junction dynamics and paracellular permeability during leukocyte activation. Recently, we reported the discovery of a new post-translational modification pathway, dhhc21-mediated protein palmitoylation, in microvascular leakage and leukocyte-endothelium interactions following infection and sterile injury. Built on these exciting findings, our program continues to advance by exploring novel diagnostic/therapeutic targets with mechanistic insights that will transform the paradigm of inflammation. Current efforts are directed to the characterization of neutrophil extracellular traps, histones and microvesicles, focusing on their cell-specific mechanisms of generation and function in the microcirculation. Studies are on-going to test the roles of palmitoylation in vesicle biogenesis, cargo composition and interaction with endothelial cells. The barrier-disrupting effects of these factors will be uncovered with in-depth molecular details on endothelial glycocalyx receptors, intracellular signal transduction, and post-translational modification (palmitoylation) of junction structures. We use a multifaceted approach that incorporates innovative molecular biology and imaging techniques (many developed in our lab) into functional analyses of vascular permeability under clinically relevant conditions. Complementary in vitro, ex vivo, and in vivo experiments are designed testing pharmacological activators and inhibitors, molecular manipulations, and genetic/chimeric alterations at cell-tissue-body levels. A unique aspect of our program lies in the translational impact achieved through the studies with intact functionally viable human organs.